Molecular biology advances in the last decade gave great promise for the introduction of new, sensitive technologies to identify various analytes in test specimens, including the ability to diagnose cancer, infectious agents and inherited diseases. Clinical molecular diagnostics depend almost exclusively on restriction enzyme analyses and nucleic acid hybridization (Southern and Northern blots) (Meselson and Yuan, 1968, Southern, 1975). Clinical tests based on molecular biology technology are more specific than conventional immunoassay procedures and can discriminate between genetic determinants of two closely related organisms. With their high specificity, nucleic acid procedures are very important tools of molecular pathology. However, nucleic acid procedures have limitations, the most important of which are the procedures consume time, they are labor intensive, and have low sensitivity (Nakamura 1993).
The subsequent attachment of radioactive or fluorescent reporting molecules to probes increased the sensitivity of nucleic acid technology. A signal from a single hybridization event could be amplified several hundred fold. The nature of the signal allowed simple visualization of each probe-target complex. However, nucleic acid reaction kinetics dictate that only 1-5% of the target molecules in a molecular pool of specimen are available for hybridization. Thus, there must be at least 5-10.times.10.sup.6 cells (or 5-10 ug of purified nucleic acid) in a specimen in order to identify affected cells with one target nucleic acid molecule in each specimen.
Another approach used to increase test sensitivity is to amplify target molecules. This was achieved by direct PCR and other target amplification methods (Saiki et al, 1985, Mullis et al., 1986). However, the use of PCR technology has been limited mainly to research applications and to use in a few sophisticated clinical reference laboratories. The power and sensitivity of PCR creates its major limitation--non-specific and false-positive amplification.
False amplification of sequence-markers for mutant oncogenes, chromosomal rearrangements or viral infectious pathogens in a clinical specimen may have profound implications for patients undergoing therapy as a result of the test results. It is technically difficult to distinguish a truly positive PCR product from the products created by non-specific amplification or by contamination with exogenous or previously-amplified DNA. There are several approaches to reducing the occurrence of false positive signals. (Saiki, 1990, Rys and Persing, 1993), however, these approaches have not brought PCR methods to the clinical laboratory.
False negative results represent another limitation of PCR-based methods. False negative results may occur due to possible inhibitory effects of the specimen on the reaction, nonoptimal concentrations of the components, or enzymes in a reaction mixture (Qi An et al., 1995). False negative results have serious implications for individuals, who in reliance on the test results do not receive timely therapy.
Amplification systems based on an enzyme of the Q-beta bacteriophage have been proposed. The Q-beta bacteriophage contains a plus-strand RNA. The plus-strand RNA serves as both mRNA for viral protein synthesis and a template for an RNA-dependent RNA polymerase, Q-beta replicase. In the presence of the plus-strand RNA, and other cofactors, the enzyme Q-beta replicase synthesizes the complementary minus-strand RNA. The minus-strand, in a turn, can serve as a template for plus-strand RNA synthesis. There is potential to amplify RNA molecules exponentially in the interaction between Q-beta replicase and its two templates--the plus and minus RNA molecules (Weissmann et al., 1968, Dobkin et al., 1979).
A number of strategies have been devised to use Q-beta native RNA templates to propagate heterologous RNA inserts (Fernandez A., 1991, Munishkin et al., 1991, Wu et al., 1992). Recombinants that form more-stable two- and three-dimensional intramolecular structures have decreased tendency to form extended plus-minus RNA--RNA duplexes during replication, which results in increased synthesis of new RNA strands (Priano et al., 1987).
Chimeric Q-beta RNA template molecules with inserts containing heterologous sequences combine two functions that make them unique among other nucleic acid molecules and position them into a special group--they can serve simultaneously as both probes and reporting molecules. As a nucleic acid fragment with a specific nucleotide sequence, the insert retains its conventional hybridization ability with high specificity to its complementary target molecules, while the whole molecule maintains its tremendous efficacy for replication. Only a few target molecules are required for detection to be amplified a hundred billion times. Theoretically, a single DNA target molecule can be detected by Q-beta replicase, which can use a single MDV-I chimeric molecule as a template to produce a detectable signal after less than one hour (Lizardi et al., 1988, Pritchard and Stefano, 1990).
Being very sensitive by nature, the methods based in Q-beta replicase technology are subject to various sources of potential contamination. The major problem of "false positives" in Q-beta replicase experiments is from the template molecules, which do not hybridize to the target nucleic acid but are still present in the reaction as contaminant-templates for Q-beta replicase.
One strategy to eliminate background created by probes that do not hybridize to the target and has been described independently in two publications (Martinelli et al., 1995, Tyagi et al., 1996). Martinelli and Tyagi propose that a chimeric template, MDV-I RNA with inserted probe sequences, be divided into halves. Each half is composed of sequences homologous to the target molecule (probes) and half of the template molecule. Neither of these halves can be amplified separately because neither of them contains the full template complement necessary for replication. The probes are designed in such a way that upon hybridization to the target molecule, they are positioned adjacent to each other, binding to the target. The terminal nucleotides of these `binary probes` are ligated by DNA ligase and the restored template-reporter can be amplified by Q-beta replicase. This strategy was applied to MDV-I DNA (Martinelli et al., 1995) and later with MDV-I RNA as a template (Tyagi et al., 1996).
One of the major advantages that differentiates Q-beta amplification assays from PCR is that Q-beta can amplify a single reported MDV-I molecule up to a hundred billion fold isothermally in less than one hour. An easily manageable, extremely sensitive and highly specific diagnostic method, Q-beta replicase assays have the same flaws common to all other methods of targeting nucleic acid molecules. None of them can be applied to proteins.
In any sample, the number of protein molecules of one kind is usually several times higher than the corresponding mRNA, and several hundred times higher than the number of genes encoding them. Using antigen-specific antibodies is a routine procedure in the modern diagnostic industry, although antibody development and purification usually require laborious work. The specificity of tests based on monoclonal antibodies depends on the capacity of antibodies to differentiate between antigens, and might approach the specificity of tests based on nucleic acid hybridization. The sensitivity of these tests, however, is routinely significantly lower than tests based on nucleic acid hybridization, even though the number of protein target molecules in each cell is relatively higher than the nucleic acid molecules corresponding to them. It is desirable to use proteins as the targets in diagnostic tests because of their abundance.
Thus, a need exists for improved diagnostic and analytical methods to detect the presence or absence of target molecules. A need also exists to detect non-nucleic acid analytes with nucleic acid chemistry.
A new technology, SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Tuerk and Gold, 1990) is a powerful tool to identify nucleic acid ligands with the ability to bind to compounds of various chemical compositions, including important medicinal targets. Similar to monoclonal antibodies, these ligands, resulting from `in vitro selection` demonstrate high affinity and specificity with various compounds, including proteins (Ellington and Szostak, 1990).
The starting point for the SELEX process is a pool of nucleic acid molecules with defined sequences into which another oligonucleotide from a library of randomized sequences is embedded. The SELEX procedure involves several cycles, each of which comprises affinity selection of the oligonucleotide from a heterogeneous population of nucleic acids (RNA or DNA) by target analyte, partition of the annealed nucleic acid and target, and amplification of the high affinity oligonucleotide subset. Each species of randomized nucleotides in the original library demonstrates a different degree of proclivity to the target molecule as a result of their sequence-dependent tertiary structure. The confiormational variability among members of such a library make it possible for the target molecule to select the RNA (or DNA) ligand with the highest affinity, termed aptamer (from Lat. `aptus`--to fit ), even though the fraction of functional molecules in the original library is very small. Positive selection through a number of cycles will progress toward the molecule with highest affinity to the target analyte and will reduce background. The number of cycles used depends upon the frequency of the `winner` aptamer in the pool of oligonucleotides at the end of each cycle, and varies from 3 to 24 with an average 10-12, depending on the nature of the target and composition of the original library (Gold et al., 1995). Twenty-five fully randomized four nucleotides (4.sup.25 =10.sup.15 species) is the practical limit of saturation in a SELEX process. The original complexity of the pool of randomized nucleotides generated is ample, considering that the number of antibodies produced by mice is five orders lower (Klug and Famulok, 1994).
As a result of `in vitro selection`, as few as three winners and as many as fourteen `aptamer-winners` are SELEXed at the end of the experiments for different targets (Gold et al., 1995). High affinity oligonucleotides have already been identified for more than forty different compounds. In general, aptamers from in vitro selection resemble protein antibodies to a great extent; they are highly specific for various antigens and more than one `winner` specific for different epitopes can be generated for the same target molecule. Their affinities reach the K.sub.d level as low as 1.0 nM (Schneider et al., 1995) or even nanomolar fractions (Bock et al., 1992, Kubik et al., 1994), which make them as good as or even better than proteinous antibodies. The aptamers make contact with the target through the domain of 10-15 nucleotides in the region of the 300-400 A.sup.2, which is approximately equal to the antigen recognition region of the Fab fragment of antibodies (Gold et al., 1995). Analogously, nucleic acid ligands with high affinity to the target analytes are termed "nucleic acid antibodies`. Similar to protein antibodies, nucleic acid antibodies can be modified in a such a way as to become carriers of a drug or other small molecule to the targets, making them a new, useful tool in therapeutic medicine, and they can be protected from endonucleases when they are used for therapeutic purposes or in diagnostic procedures on easy-to-obtain human blood or urine specimens (Pieken et al., 1991).
One of SELEX's major limitations, besides being technically very laborious and time consuming, follows from the conformational complexity of winning aptamers which determines their high affinity to the target analyte. The complex tertiary structure of the aptamers may affect the outcome of amplification performed by PCR or during cDNA synthesis using reverse transcriptase enzyme as the component of each cycle. Molecules with less complex structures have a comparative advantage over the most functionally potent aptamers because oligomers with `simple` structure replicate more efficiently (Klug and Famulok, 1994).